1. Api 20E

2. principle
The API 20E System is a standardized, miniaturized microtube system consisting of 21 conventional “basic” and 6 supplementary biochemical tests used for the identification of Enterobacteriaceae and other non-fastidious Gram-negative bacteria.
The API 20E System consists of microtubes containing dehydrated substrates. The substrates are reconstituted by adding a bacterial suspension.
They are then incubated so the organisms react with the contents of the tubes. Finally, they are then read when the various indicator systems are affected by the metabolites or added reagents, generally after 18 to 24 hours incubation at °C. Refer to API 20E package insert for the principles involved in each of the reactions, reactive ingredients and quantity of ingredients in each tube.
Cupule
tube

3. Biochemistry A)Basic tests
ONPG : ortho-nitrophenyl b-D-galactopyranoside.
ONPG is a substrate that have B-galactosidic bond “like lactose” but didn’t need permease to enter bacterial cell.
ONPG “colourless” galactose + ortho-nitrophenol “yellow/pale yellow”
B-galactosidase
Lac operon
B-alactosidase
Permease
B-trans-acetylase
Latose fermenter ++
Delayed fermenter
+/-
Non-fermenter -
+ -

4. Biochemistry + - This microtube contains phenole red indicator
ADH : arginine Dihydrolase
argenine citrullin + NH3
citrullin + phosphate ion carbamoil phosphate + ornithine
carbamoil phosphate ATP + CO2 + NH3
This microtube contains phenole red indicator
orange color consider +ve only in first 24h
ADH
Catabolic arginine carbomoly transferase
Carbamoil kinase
+ -

5. Biochemistry + - + - LDC : lysine decarboxylase. “as LIA”
Lysine cadaverine (pH ) +CO2
ODC : ornithine decarboxylase. “as MIO”
ornithine putriscine (pH ) +CO2
LDC
Phenole red
+ -
ODC
Phenole red
+ -

6. Biochemistry + - Na +CO2+H2O Na2CO3 CO2+pyruvate
CIT : testing the ability of bacteria to utilize citrate as a carbon source
sodium citrate acetic acid + oxaloacetic acid
Na +CO2+H2O Na2CO CO2+pyruvate
ammonium salt NH3+NH4OH
bromothymol blue : yellow (6) > green (6.9) > blue (7.6)
Citrate layase
OAA
decarboxylase
+ -

7. Biochemistry + - + - H2S : H2S production test.
sodium thiosulfate H2S (g)
H2S ferrous sulfide “black ppt”
URE : urease utilization test.
urea CO2 + NH3
urease works at both aerobic and anaerobic
conditions, but it prefers the second.
Reduction “H+”
Ferrous sulfate
+ -
urease
Phenol red
+ -

8. Biochemistry + - TDA : tryptophan deaminase.
tryptophan indole-pyruvic acid “form within 24 h” + NH3
indole-pyruvic acid +10%FeCl3 + HCl + hydrazine ferric hydrazon
HCl added to TDA reagent to break dow the product of the reaction between FeCl3 and tryptophan to prevent false positive rasults.
TDA
“” Reddish brown ppt ””
+ -

9. Biochemistry + - + - IND : tryptophan utilization test.
tryptophan indole + pyruvic acid + NH3
indole pink or red “with or without ring”
VP : Voges Proskauer test.
sodium pyruvate 2,3- Butanediol
acetoin red color “within 10 min”
arrangement of reagents application is not necessary
because of the absence of peptone
tryptophanase
Kovac’s reagent
Isoamyl alcohol + HCl + para-nitrophenyl aminobenzaldehyde
+ -
Barrit’s reagent + creatine
VP “1” : 40% KOH
VP ”2” : 5% alpha-naphthol
+ -

10. Biochemistry + - + - GEL : gelatin liquefaction test.
gelatin + cool chart black ppt
12-20 : sugar fermentation/oxidation tests.
sugar fermentation or oxidation
acid product
+ve “yellow”
21. Oxidase test : performed on external fresh culture.
gelatinase
+ - +
Bromothymol blue -

11. note
Fermentation (Enterobacteriaceae, Aeromonas, Vibrio) 1. Fermentation of the carbohydrates begins in the most anaerobic portion (bottom) of the tube. Therefore, these reactions should be read from the bottom of the tube to the top. 2. A yellow colour at the bottom of the tube only indicates a weak or delayed positive reaction. Oxidation (Other Gram-negatives) 1. Oxidative utilization of the carbohydrates begins in the most aerobic portion (top) of the tube. Therefore, these reactions should be read from the top to the bottom of the tube. 2. A Yellow color in the upper portion and a blue color in the bottom portion of the tube indicate oxidative utilization of the sugar. This reaction should be considered positive only for non-Enterobacteriaceae Gram-negative rods. This is a negative reaction for fermentative organisms such as Enterobacteriaceae.

12. Biochemistry B)Supplementary tests “needed only with multitaxon code”
NO2
Reduction of nitrate to nitrite only
Before addition of reagents (sulfanillic acid & α-naphthyl amine), observe GLU tube (positive or negative) for bubbles. Bubbles are indicative of reduction of nitrate to the nitrogenous (N2) state.
A positive reaction may take 2-3 minutes for the red color to appear.
Confirm a negative test by adding zinc dust. A pink-orange color after 10 minutes confirms a negative reaction. A yellow color indicates reduction of nitrates to the nitrogenous (N2) state.
N2 Gas
Complete reduction of nitrate to N2 gas or amines
MOB
Observation of motility by semisolid agar
McC
Growth on MacConkey agar
OF-O
Oxidative utilization of glucose (OF-open)
OF-F
Fermentative utilization of glucose (OF-closed)
oxidation test tube
fermentation test tube
1% glucose
Nutrient agar
Bromothymol blue
1% glucose
Nutrient agar
Bromothymol blue
Sterile mineral oil

13. Biochemistry B)Supplementary tests
Note: if these tests used, you have to delay the reading of all result for 48h

14. material API 20 E Strips Incubation boxes “ tray and lids”
Report sheets
Sterile syringe and needle
Disposable plastic inoculating loop
5 ml sterile normal saline
Sterile Mineral Oil
MacConkey agar plate

15. procedure

16. procedure

17. procedure

18. procedure

19. procedure

20. procedure

21. Interpretation
On the report sheet, the test are separated into groups of three and number 1 , 2 or 4 is allocated for each test. By adding the numbers corresponding to the positive reaction within each group, a7- digit profile number is obtained for 20 tests of the API 20E strip.
The 7- digit profile is then compared with the numerical profile in the API 20 E analytical profile index book to obtain the organism identification.

22. Exercise: find the code

23. Thank you